The present invention provides a means for immobilizing proteins through covalent attachment to inorganic oxide surfaces via thioester-containing coupling chains. Once immobilized, the protein can be released from the surface by cleavage of the coupling chains at the thioester linkage.
Covalent coupling of proteins to solid supports generally relies on common organic reactions. Most of the methods which have been used were reviewed by Mosbach, K., Ed. (1976), Methods Enzymol. 44, Academic Press, New York or by Zaborsky, O. (1973), Immobilized Enzymes, CRC Press, Cleveland, Ohio.
Generally, characterization of immobilized enzymes has been limited to kinetic studies or to examination of fluorescent spectra (see Gable, D., Steinberg, I. Z., and Katchalski, E. (1971), Biochemistry 10, 4661-4669; Horton, H. R., and Swaisgood, H. E. (1976), Methods Enzymol. 44, 516-526; and Swaisgood, H. E., Janolino, V. G., and Horton, H. R. (1978), Arch Biochem. Biophys. 191, 259-268). This restriction severely limits the application of immobilization techniques for studies of the reformation of the tertiary structure or quaternary structure in proteins. However, the capability of selectively releasing an immobilized protein following various experimental operations would allow the application of many additional biochemical techniques for examination of structural features.
One of the problems often encountered in studies of immobilized enzymes is elucidation of the factors involved in altered kinetic patterns. The catalytic rates of enzymes may be affected in several ways by immobilization: (1) by changes in specific rate constants as a result of conformational changes in the enzyme's structure per se, alteration of its micro-environment, or steric hindrance of substrate access; (2) by partitioning of solute molecules (including substrates and products) due to specific interactions with the matrix; and (3) by diffusion inhibition (see Engasser, J. M., and Horvath, C. (1973). J. Theor. Biol. 42, 137-155; Goldstein, L. (1976), Methods Enzymol. 44, 397-443; Kobayashi, T., and Laidler, K. J. (1973), Biochim. Biophys. Acta 302, 1-12 and Cho, I. C., and Swaisgood, H. (1974), Biochim. Biophys. Acta 334, 243-256). It has been very difficult to distinguish among these various kinetic effects while an enzyme remains immobilized.
Removal of an immobilized enzyme from its supporting matrix in such a way as to retain a portion of the chain involved in the previous covalent attachment would provide a feasible means for investigation of conformational changes or steric hindrance directly related to the immobilization procedure. Also, the effect of the microenvironment of the matrix surface, itself, could be thus eliminated, so that the effects of partitioning and diffusion can be clearly separated experimentally from intrinsic changes in specific rate constants.
The possibility of releasing protein immobilized through an azo linkage with sodium dithionite or of releasing thioester-linked protein with hydroxylamine or high pH had been suggested by Cuatrecasas, P. (1970a), Nature 228, 1327-1328. More recently, Chan, W. W. C., and Mosbach, K. (1976), Biochemistry 15, 4215-4222 have reported a procedure for reversible immobilization based on a disulfide linkage. An attempt to form a selectively cleavable covalent bond, by incorporating a thioester linkage, was reported by Brown, J. C., and Horton, H. R. (1973), Fed. Proc. 32, 496. Carbodiimide-activated succinylated glass beads (Brown, J. C., Swaisgood, H. E., and Horton, H. R. (1972), Biochem. Biophys. Res. Commun. 48, 1068-1073) were treated with 2-mercaptoacetic acid; the derivatized glass was then treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and finally exposed to the protein-containing solution. Subsequently, immobilized protein could be removed from the glass surface by cleaving the thioester bonds with hydroxylamine, but the yield of released protein was low (approximately 15% of that immobilized).
Thus, many methods for covalent immobilization of biochemicals have been described as noted above. However, for most of these methods the immobilized molecule, especially enzymes or proteins, can not be released without destruction of its integrity. The only reported method for reversible covalent attachment of molecules to surfaces involve linkage through disulfide bonds. However, these linkages would not be stable in the presence of mild reducing agents. Such agents are often required for the maintenance of biological activity.
An ideal method for reversible covalent immobilization would have the following characteristics:
1. The activated form of the particles used for immobilization should be very stable so that such particles could be shipped and stored until immobilization of a biochemical is desired. PA1 2. The immobilization should be accomplished simply by addition a solution containing the biochemical. PA1 3. The chemistry of immobilization should not affect the biological activity of the molecule. PA1 4. The immobilization method should achieve maximum loading of the particle surface area with the biochemical. PA1 5. The covalent chain linking the biochemical to the surface should be stable under most conditions so that the biochemical is not slowly leached from the surface. PA1 6. The covalent chain linking the biochemical to the particle surface should be cleaved by specific reagents under very mild conditions so as not to alter the biological activity of the released biochemical. PA1 a. treating an inorganic oxide material to provide an activated surface thereon; and PA1 b. contacting the activated surface with protein having a reactive amino group whereby said protein is covalently bonded to the activated surface. PA1 a. silanizing and succinylating an inorganic oxide material to provide a succinamidopropyl-surface; PA1 b. converting the succinamidopropyl-surface to the acyl chloride derivative by treatment under anhydrous conditions; PA1 c. reacting the acyl chloride derivative with either 3-mercaptopropionic acid or mercaptoacetic acid under anhydrous conditions whereby the surface of the material is activated; and PA1 d. drying the activated material.
Accordingly, it is the primary object of the present invention to provide a method for the immobilization of proteins which to a large degree meets the ideal immobilization procedure noted above.
It is a further object of the present invention to provide a relatively simple method for immobilization of proteins while at the same time achieving a high degree of loading and biological activity of the immobilized protein.
These and other objects of the present invention will become apparent from the discussion which follows.